Muscle contraction is regulated by interactions of free cytosolic calcium with calcium-activated proteins such as troponin C or calmodulin. These proteins initiate a cascade of events leading to cell shortening and muscle contraction. SERCA is a membrane protein that terminates or prevents the muscle contraction. This protein transfers cytosolic calcium against the concentration gradient into the sarcoplasmic reticulum (SR) and is present in many tissue and development-specific isoforms. Cancer is a disease that in many cases confers resistance to apoptosis. SERCA can also transfer cytosolic calcium against the concentration gradient into the endoplasmic reticulum, thereby modulating the intracellular calcium concentration and reversing resistance to apoptosis in cancer cells.
PLB is a small membrane protein that inhibits SERCA at submicromolar calcium concentration. The inhibition of SERCA by PLB can be relieved either by phosphorylation of PLB or by elevation of calcium concentration to the micromolar range. Through its inhibitory effects on SERCA, PLB represses both the rates of relaxation and contraction of the muscle cells. Because SERCA and PLB play a major role in muscle contraction, abnormal SERCA or PLB activity as well as SERCA-PLB interactions have been implicated in a wide variety of muscle diseases. For example, enhanced inhibition of SERCA by PLB has been associated with heart failure. (Delling et al., (2000) Nature Medicine 6:942-943; Kiriazis et al., (2000) Annu. Rev. Physiol. 62:321-351). The R9C mutation of PLB in humans was directly linked to development of dilated cardiomyopathy and progression to heart failure in young adults (Schmitt et al., (2003) Science 299, 1410-1413).
Certain molecules that modulate the SERCA-PLB complex or its microenvironment bind SERCA directly, even in the absence of PLB. In several different cell types, excessive cytosolic calcium has been shown to induce apotosis. Id.; see also Nicotera et al., Cell Calcium (1998) 23: 173-180. For example, Christensen et al. reported that the growth of LNCaP prostate cancer cells in mice was inhibited by administration of thapsigargin, which is a SERCA inhibitor. Bioorg. Med. Chem. 14 (2006) 2810-2815, incorporated herein by reference in its entirety. The authors attributed this observed inhibition to the potency of thapsigargin as an inhibitor of SERCA. Id. at 2810. Specifically, the authors explained that SERCA inhibition induces an increase in cytosolic calcium concentration, which eventually causes apoptosis. Id. In another example, cyclooxygenase 2 (COX-2) inhibitors, such as celecoxib, have emerged as potential anticancer agents. Schönthal, Cancer Lett. (2008), doi: 10.1016/j.canlet.2008.07.005, the entirety of which is incorporated herein by reference. It is thought that the inhibition of COX-2 is not involved in the anticancer effect of celecoxib; rather, celcoxib inhibits tumor growth via inhibition of SERCA. Id. at 4-5. Schönthal is now taking advantage of this discovery by designing and synthesizing new compounds that even more effectively target SERCA, which may lead to the discovery of anticancer agents. Id. Based on theses findings, it is contemplated that molecules capable of regulating SERCA, the SERCA-PLB complex or its microenvironment would have broad applications for treating SERCA- or PLB-related diseases.
Therefore, a consistent and reliable method of identifying molecules that modulate SERCA, the SERCA-PLB complex or the microenvironment of the complex would merit further investigation. Due to the complexity of membrane proteins such as SERCA and PLB, it has been very difficult to produce a synthetic system that can recapitulate the cellular interactions in a large-scale reproducible manner. Moreover, observation of modulated SERCA-PLB complex or its microenvironment has been limited, for example, by unsuitable FRET potency or strong SERCA and PLB interactions that cannot be easily disrupted. Thus, a HTS assay that is capable of performing said method in a membrane would be valuable in clinical and pharmaceutical research for SERCA or PLB-related diseases.